COX2: A Prognostic Inflammogenic Marker Drives Cervical Carcinogenesis In Vivo Through NFκB/IAP/p53Axis

doi:10.21203/rs.3.rs-814913/v1

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COX2: A Prognostic Inflammogenic Marker Drives Cervical Carcinogenesis In Vivo Through NFκB/IAP/p53Axis

https://doi.org/10.21203/rs.3.rs-814913/v1

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Cycloxygenase2, a prostaglandin synthesizing enzyme is a key player in inflammation-induced vasculogenesis that enables tumor growth. This study explores the central role of COX2 and its relative prosurvival proteins in evoking inflammatory events during development of an in vivo cervical cancer model upon chronic treatment with 3-methylcholanthrene (3MC; a chemical carcinogen) in virgin-female Swiss Albino mice. Chronic painting of mice cervix with 3MC solution triggered the persistent expression and activity of COX2; eventuating in overexpression of major prosurvival molecules (NFκB, XIAP, survivin, GM-CSF1) and proliferative antigens (Ki67, PCNA). COX2-arbitrated prosurvival signaling subsequently deranged the expression profiles of tumor supressor proteins (p53/Acetyl-p53, p21, Rb) within the cervix. COX2 helmed molecular alterations successively surged leukocyte influx within cervix; catering in localized inflammation which gradually distorted its tissue architecture. Cervical carcinogenesis was further braced by higher levels of systemic-ROS and RNS, escalated iNOS activity and compromised anti-oxidant enzyme capacities, which were accompanied by splenomegaly. Additionally, circulation of blood-leucocytes with damaged DNA throughout the mice body, envisaged the impact of cervix-limited inflammation upon the mice physiology. Conclusively, the present study deciphered the role of COX2 effectuated NFκB/IAP/p53 functions in sequestering the contributors of localized and systemic inflammogenesis for propelling 3MC-mediated cervical carcinogenesis in vivo.

Immunology

COX2

NFκB

IAPs

p53

inflammation

cervical cancer.

Cycloxygenase2 (COX2) is preeminent in inflammogenesis of cancers. Formally termed Prostaglandin H₂ Synthase-2 enzyme, COX2 catalyses the rate-limiting step of mitochondrial fatty acid oxidation concerning the conversion of arachidonic acid to biologically active prostanoids which are propagators of inflammation [1, 2]. These prostanoids maintain COX2 expression to activate ROS/AKT/activator protein1 [3] and switch on the kinase-cascades [4, 5] for enabling malignant transformations. Besides, COX2 promotes ‘angiogenesis’, the hallmark episode of new blood vasculature growth in solid tumors which assists them to sustain independently. Vascular Endothelial Growth Factor (VEGF), the key regulator of ‘angiogenic switch’ is also instructed by COX2 for undertaking endothelial growth [6, 7]. Therefore, researches delving into contributions of COX2 in various neoplastic progressions are on rise.

With a chromosomal location of 1q31, COX2 gene is highly inducible in nature [8] due to which it gets overtly expressed for organizing inflammatory events only upon viral oncogenic inductions, chemical carcinogenic triggers or in pathological conditions developed due to chronic viral or bacterial infections [9, 10]. In the incidences of Human Papilloma Virus (HPV) mediated cancers like cervical and oral carcinomas, extended viral latency period was found to be closely associated with COX2 generated non-compensatory inflammation states [11]. Similarly, persistent xenobiotics exposure induced COX2 activities which reportedly enhanced skin inflammations; bringing forth carcinogenesis [12]. Recent experimental reports are evidential for the prerequisite need of COX2 in cancer stem cell differentiation and proliferation [13, 14]. The Polycyclic Aromatic Hydrocarbons (PAHs) of lower molecular weight emitted along with cigarette smoke were found to effectuate p38 MAP-Kinase to cause COX2 mRNA overproduction. As a consequence, eicosanoid signalling got kick-started within the lung epithelium for launching carcinogenic changes [15]. Often in lymphomas, COX2 observably upgraded proliferative, angiogenic and invasive potentials of leukocytes [16, 17]. Multiple cancers are characterized by genetic aberrations along with polymorphisms in their COX2 gene [18–22].

Several interesting documentations are supportive of COX2 being the central under pinner of inflammation, angiogenesis and tumorigenic progression [23, 24]. COX2 abetted inflammation becomes the ‘formative-ground’ of a strong prosurvival signalling network, characterized by deteriorated tumor suppressor activities. Generally, it is a common observation that gain-of-function mutations in the effector genes of the Ras-MAPK and the PI3K/AKT-signalling pathways principally dictate over the malignant transformation processes. Reportedly, COX2 could potentiate many cancers for phenotypically mimicking the effects of these two pathways; even when the concerned driver mutations are absent [25]. In the hypoxic tumor cores, rise in expression of Hypoxia Inducible Factor-1 (HIF-1) collaterally accelerates COX2 kinetics for permitting the expressions of Nuclear Factor κB (NFκB) [26] and its effector-Inhibitor of Apoptosis Proteins (IAPs) [27]. COX2-derived prostanoids further activate MAPK pathway to allow VEGF translation; aiding in vasculogenesis [28, 29]. Additionally, COX2 adorns functionality by repressing the tumor suppressors such as p53, p21 or Rb over activation of Bcl₂, the anti-apoptotic protein [30, 31]. The relentless cytokine storm following COX2 activation relays dysregulated inflammatory signals which alter the functional differentiation of immune cells for promoting tumorigenesis [32]. COX2 and the related cytokines are held responsible for switching the developmental fate of ‘tumor-inhibiting’ M1 macrophages to ‘tumor-promoting’ M2 macrophages or Tumor Associated Macrophages (TAM) [33, 34]. Therefore, COX2 and its effectors empower a growing tumor mass with the armour of immune evasion [35]. Inevitably, COX2 is the prima facie requirement for promoting autonomous tumor growth.

With regard to the prevalent reports, the present study was designed to investigate the mediatory role of COX2 in domineering the inception and progression of cervical carcinogenesis within Swiss Albino mice upon chronic treatment with 3methylcholanthrene (3MC), a potent chemical carcinogen of PAH family. This study systematically dismantled the role of COX2 in sequestering inflammogens to drive the activation of prosurvival signaling nexus for promoting cervical carcinogenesis in vivo.

Animal Maintenance

Virgin female Swiss Albino mice (Mus musculus; 5–6 weeks old; weight: 23-25gms) obtained from the Central Animal Facility of CNCI were housed in polyvinyl cages within well-ventilated rooms under ideal conditions (temperature: 22⁰C; relative humidity: 50–60%; 12 hour day/night cycle). Prior to treatment initiation, the animals were subjected to a two-week acclimation period during which all the female and male mice were kept in complete isolation from each other. This induced pheromone influenced oestrous cycle synchrony thereby nullifying the hormonal interferences [36]. Standard guidelines laid down by the Institutional Animal Ethics Committee (IAEC) certified by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, were strictly followed for undertaking all animal experimentation.

Experimental design

Random allocation of 100 mice into three broad groups was accomplished on the basis of body weight. Each group was further classified into subgroups separated in two batches of 10 animals [No of mice (n) = 5 /cage (x2) for each subgroup]. Group I was kept as an ‘untreated’ control group where mice did not receive any treatment. Group III (groups 6–10) comprised of mice which had their cervix chronically painted with 3MC (MP Biomedicals, USA) solution prepared in Petroleum Ether (PET, Merck, Emplura, Germany) for respective intervals of 6, 12, 16, 24 and 30 weeks [37]. It was specifically designated as ‘treatment’ group for studying cervical carcinogenesis along these time spans. To rule out any carcinogenic effect of the solvent (if any), Group II (groups 1–5) was assigned as a ‘vehicle control’ batch, with mice being solely treated with PET. Food and water was given ad libitum. Mice were periodically monitored for any visible health abnormalities and deaths. This purposeful procedure of mice grouping is well-represented in Table 1.

Table 1

Animal Groups for Experimentation
Sl.No	ANIMAL GROUPS	TREATMENT APPROACH
1.	GROUP-I (Control)	Untreated group
2.	GROUP II (Vehicle Control)	Chronic treatment of mice cervix with Petroleum Ether (PE)
	Group 1	6 weeks
	Group 2	12 weeks
	Group 3	16 weeks
	Group 4	24 weeks
	Group 5	30 weeks
3.	GROUP III (Treatment)	Chronic 3MC treatment of mice cervix (Dosage- 0.6mg/ml)
	Group 6	6 weeks
	Group 7	12 weeks
	Group 8	16 weeks
	Group 9	24 weeks
	Group 10	30 weeks

After every mentioned treatment period, mice from all the three groups were sacrificed and their entire female reproductive tissue was excised out. In order to track the presence of any cervical tumor, these reproductive tissues were longitudinally opened up for examination through its upper vaginal (ectocervix) region. Cervix tissues (with or without tumor) were decapitated out for comparative studies. Besides cervix, blood, and spleen were also collected for experimental purposes.

Record of body weight and carcinogenic incidences

Weekly body weight alterations in mice were recorded to assess the impact of chronic 3MC and PET treatments on their physiology with time. An orderly record of tumor incidences along with gradually acquired cervical dysplastic stages in the 3MC treated mice batches was also maintained.

Cytopathological study

Smears of cervical exfoliated cells suspended in Phosphate Buffered Saline (PBS, pH-7.4) were fixed with 100% ethyl alcohol and stained as per the protocol of Mahapatra et al., (2020) [37]. Sequential polychromatic staining with Harris Haematoxylin (HH), Orange G6 (OG6) and Eosin Azure (EA50) [Merck Millipore, Mumbai, India] followed by excess stain removal in tap water, 70% and 90% alcohol was observed. Finally stained slides were mounted and observed under a light microscope (Zeiss). Randomly, 50 fields were microscopically scanned for the presence of significant cytopathological changes in each slide.

Enumeration of differential count of cervical leukocytes

A numerical count of the microscopically identified subpopulations of cervical leukocytes was quantitated as ‘Differential Leukocyte Count’ (DLC). Over 50 fields of Pap stained slides were randomly scanned for calculating DLC. For each field, DLC was enumerated as a percentage (%) of specific number of leukocyte subpopulations (eosinophils, neutrophils and monocytes) observed among the total number of leukocytes [DLC= (Number of specific type of leukocytes)/ (Total number of leukocytes) x 100].

Histology

Dissected cervical and spleen tissues were washed in cold normal saline (0.9%), fixed in 10% (w/v) Neutral Buffered Formalin (NBF) for 24 h, processed through alcohol grades and xylene, followed by embedding in paraffin. These paraffinized tissue blocks were cut into thin (~ 5µm) sections using a microtome and stretched over grease-free glass slides. These sections were further deparaffinised in xylene followed by successive staining in Delafield haematoxylin [DH; haematoxylin powder, 100% alcohol, (NH₄)Al(SO₄)_2, Glycerol, H₂O] and 2% Eosin. After mounting, 10–20 fields of these stained slides were examined under a light microscope (Zeiss) for documentation of the histological changes

Immunohistochemistry

Cervical tissue sections (~ 5µm thick) were stained with the primary antibodies of COX2, NFκB (p50/p65), XIAP, survivin, Ki67 and PCNA proteins for locating them within tissues by following the protocol of Basu et al., (2020) [38]. Paraffin from stretched tissue sections was removed by heating the slides at 65⁰C for 20 min followed by xylene treatment and rehydration through 100%, 90 %, 70 % and 50 % alcohol downgrades. After washing these sections serially in PBS for 10min, ‘antigen-retrieval’ was carried out using pre-heated citrate buffer [pH-6; comprising of C₆H₈0₇.H₂O and (CH₂COONa)₂.2H₂O] at 85⁰C for 1 h following which they were incubated with the respective primary antibodies diluted in 1 % bovine serum albumin (BSA; Sigma Aldrich, USA) solution within a humid chamber overnight at 4⁰C. Excess primary antibodies were washed in 1X PBS. Slides were further incubated with HRP conjugated secondary antibodies (1:500) in 1% BSA solution for 2 h at 37⁰C followed by immunostaining with the chromogenic substrate 3–3′ diaminobenzidine (DAB; SantaCruz Biotechnology, USA) and counterstaining with DH. These slides were dehydrated through successive alcohol grades and xylene. Finally, they were mounted in DPX for observation under light microscope (Zeiss). About ten fields were scanned in order to score for the positive staining intensities. Staining intensities (1 = weak, 2 = moderate, 3 = strong) were enumerated as per the percentage of positively stained cells (< 1 = 0, 1–20 = 1, 20–50 = 2, 50–80 = 3 and > 80 = 4). Final evaluation of the tissue specific protein expressions was made as low (Score 0–2), intermediate (2–5), and high (Score 5–7) levels.

The primary antibodies employed were: COX2 (GeneTex, 1:1000), NFκB(p50/p65) (GeneTex, 1:1000), Ki67 (GeneTex, 1:1000), PCNA (GeneTex, 1:1000), XIAP (GeneTex, 1:1000) and survivin (GeneTex, 1:1000).

Preparation of tissue lysates

Cervix tissues along with the adjoining tumor regions were dissected out, washed, and pooled separately from Group I, II and III mice. Tissue and tumor parts were dried, weighed and homogenized in Radio-Immunoprecipitation Assay Lysis buffer (RIPA;pH-8 comprising of 5M NaCl, 0.5M EDTA,1M Tris, NP-40,10% Sodiumdeoxycholate,10% SDS). The extracts were kept in ice for 30 min followed by sonication and centrifugation at 10,000g for 20 min at 4⁰C. The resulting supernatants were stored in chilled vials at -20⁰C.

Estimation of total protein

Total protein content of the tissue extracts was spectrophotometrically (VARIAN) estimated using 1X Bradford’s reagent (HIMEDIA, PA, USA) against a standard curve of BSA. Absorbance was recorded at 595 nm with the experiment being repeated for 5 times.

Western Blot Analysis

The expression statuses of inflammatory mediators (COX2, GM-CSF1), tumor suppressor proteins [p53, acetylated-p53 (lys373), p21, Rb], prosurvival molecules (NFκB, XIAP, survivin) and proliferative antigen (Ki67) were comparatively studied by western blotting. Equitable amounts of cervical protein were respectively loaded into each well of SDS-polyacrylamide (SRL, Mumbai, India) gels, electrophoretically separated using electrophoresis buffer (25mM Tris, 192mM glycine, 10% SDS) and electro-transferred to nitrocellulose membranes with the aid of a transfer buffer (250mM Tris, 192mM glycine, 10% Methanol). These membranes were blocked with 5% (w/v) BSA solution, washed with Tris Buffered Saline (TBS; pH-7.5; 25mM Tris.HCl, 150mM NaCl) and incubated overnight with primary antibodies at 4⁰C under constant shaking. Blots were thereafter washed with TBST Buffer solution (TBS; tween 20) for 4 times and subsequently incubated with alkaline phosphatase conjugated secondary antibodies (GeneTex, 1:500dilutions in TBS) at 4⁰C for 2h, followed by TBST washing (4times) and incubation with the chromogenic substrate 5-bromo, 4-chloro, 3-indoylphosphate/ Nitro-Blue tetrazolium (BCIP/NBT; SantaCruz Biotechnology, USA) for visualizing protein expressions in the form of bands. β-actin was used as a loading control protein. These experiments were performed in triplicate.

The primary antibodies used were: COX2 (GeneTex, 1:1000), GM-CSF1 (Santa Cruz, 1:1000), NFκB(p50/p65) (GeneTex, 1:1000), Ki67 (GeneTex, 1:1000), PCNA (GeneTex, 1:1000), XIAP (GeneTex, 1:1000), survivin (GeneTex, 1:1000), Acetyl p53(lys 373) (Merck-Millipore, 1:1000), p53, p21, and Rb (Santa Cruz, 1:1000) and β-actin (GeneTex, 1:1000).

Quantitative estimation of COX2 activity

COX2 enzyme (E.C 1.14.99.1) was spectrophotometrically quantitated at 590 nm using COX activity assay kit (Cayman Chemical, Cat No: 760151 Ann Arbor, MI, USA) as per the provided protocols. Results were represented in nmol/min/ml (U/mg of protein).

Isolation of blood leukocytes

One volume of mice blood collected aseptically from heart was mixed with three volumes of Solution A (pH-7.2; 0.87%NH₄Cl in 10mM Tris HCl), ice incubated for 20 min and centrifuged at 400g for 20 min at 0⁰C. The supernatant was discarded and the pellets were again resuspended in Solution A followed by centrifugation at 400g for another 20 min at 0⁰C. Resulting pellets were suspended in Solution B (pH-7.2;0.25M mesoinositol, 10MmNa₂SO₄, 1Mm MgCl₂), cold centrifuged at 1500 rpm for 5 min at 4⁰C succeeded by re-suspension of pellets in HEPES Buffered Saline (HBS; pH 7.4;140 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose). Cell numbers were adjusted as per experimental requirement.

ROS generation

Reactive Oxygen Species (ROS) generated due to chronic treatment with 3MC was quantitated as per the principle of Sinha and Roy (2010) [39]. Isolated leukocytes (10⁶cells/ml/point) suspended in HBS were incubated with a fluorescent probe, 2’,7’-dichlorofluorescein dihydroacetate or DCFH-DA (10µM, Sigma-Aldrich, USA) for 45 min at room temperature in complete darkness. DCFH-DA passively diffuses within cells to transform into a diol moiety which is further oxidized into a fluorescent compound, 2’, 7’-dichlorofluorescein (DCF) by intracellular ROS. DCF was quantitated spectrofluorimetrically (VARIAN; Excitation: 485 nm and Emission: 530 nm). For each point, readings were recorded for about five times in triplicate attempts of the experiment.

RNS generation

Equal volumes of peritoneal macrophage suspension (10⁶cells/ml) in PBS and Griess reagent (1% sulphanilamide, 0.1% naphthyl ethylenediamine hydrochloride and 5% orthophosphoric acid) was incubated at 37⁰C for 30 min in a humidified chamber for quantifying Reactive Nitrogen Species (RNS) levels [40]. Absorbance was recorded at 550 nm with a spectrophotometer (VARIAN) against a standard blank. NO levels were enumerated against a standard curve of sodium nitrite. Readings for each point were taken for about five times in triplicate attempts.

Quantitative estimation of nitric oxide synthetase activity

Indirect assessment of Nitric Oxide synthetase (iNOS) enzyme (E.C1.14.13.39) activity was done using a spectrophotometer by calculating the percentage of L-citrulline catalytically produced from L-arginine by means of NOS activity Assay Kit (Cayman Chemical, Cat No: 781001, Ann Arbor, MI, USA) in accordance with the kit instructions.

Antioxidant Enzyme Activity

Free Radical quenching capacity of the antioxidant scavengers present in the blood serum isolated using serum separating vials, was assessed with the help of Antioxidant Assay Kit (Cayman Chemical, Cat No: 709001, Ann Arbor, MI, USA). Enzyme activity was expressed as IU/L with the help of a spectrophotometer where readings were recorded within the absorbance range of 405-750nm.

Single cell gel electrophoresis (SCGE or Comet assay)

The clastogenic effect of 3MC on DNA was assessed following the standard laboratory protocol [41]. Concisely, a suspension of 0.6% (w/v) low melting agarose (LMA; Sigma-Aldrich, USA) and isolated leukocytes (1x10⁴cells) was smeared over a frosted microscopic glass slide which was priorly coated with the fixative 0.75% (w/v) normal melting agarose (NMA; Lonza, USA). Following solidification at 4⁰C, cell and nuclear membranes were lysed in lysis buffer (pH-10; 2.5 M NaCl, 0.1 M Na₂EDTA, 10 mM Tris, 0.3 M NaOH, 1%Triton X-100, and 10%DMSO). Exposed DNA from the lysed out leukocytes were unwound in a highly alkaline electrophoresis buffer (pH > 13.0; 300 mM NaOH, I mM Na₂EDTA) prior to electrophoresis for 20 min (300 mA, 20 V). Slides were washed in neutralizing buffer (Tris 0.4 M, pH 7.5) thrice, stained with ethidium bromide (final concentration 40 µg/ml) and examined under a fluorescence microscope (Leica). Image analysis, head DNA quantification, comet tail DNA length estimation and comet tail moment calculation were performed using Komet Software.

Statistical Analysis

The mean values of the control, vehicle control and treatment mice groups were compared by factorial Analysis of Variance (ANOVA). The relationship between the studied parameters was analysed by calculating Pearson’s correlation coefficient using CORREL function of Microsoft Excel. Data were expressed as mean ± standard deviation (S.D.). p value calculations were performed using Prism GraphPad Software. ^*p < 0.005 and ^**p < 0.01 were considered statistically significant in comparison to control batches.

The optimum 3MC dose of 0.6 mg/ml was selected as the safest and efficacious treatment dose (< LD50) only after checking its impact on in vivo toxicity parameters namely SGPT, SGOT, ALP and creatinine levels (data not shown). Carcinogen preparation process is elaborated in Fig. 1A. The treatment durations of 6, 12, 16, 24 and 30 weeks were also accordingly identified. A clear schematic outline of the experimental design with appropriate timeline of animal sacrifice has been illustrated in Fig. 1B.

Emergence of tumor at the cervical region upon chronic 3MC treatment

Prior to sacrifice, the experimental mice were preliminarily monitored for any apparent changes in the appearance of their orifice and internal female reproductive organ owing to chronic treatment with PET or 3MC. Relevant observations noted among mice of Group-I, II and III are comparatively depicted in Fig. 2A and 2B. Obvious progressive changes in the vaginal orifice of 3MC treated mice (Fig. 2Axi-xv) were an outcome of internal changes in their reproductive system (Fig. 2B). As indicated in Fig. 2Biv and v, remarkable alterations of the mice reproductive organs in 12 and 16 weeks of 3MC treatment mostly included mild thickening along with growth of new blood vasculature. Group-III mice from 16th week batches had a mildly swelled orifice (Fig. 2Axiii; indicated by arrow). Further extending 3MC treatment beyond 24 weeks resulted in tumor growth at the cervical region (Fig. 2Bvi) leading to explicit swelling of the lower abdominal area (Fig. 2Axiv & xv; indicated by arrows). No such abnormalities were noted in the vehicle control and control mice neither amidst the treatment intervals nor after 24 weeks.

Cytopathological changes in terms of increased ‘keratinisation’ of the cervical squamous epithelial cells bearing ‘nuclear holes’ (Fig. 2Cxii; indicated by arrows) became perceptible amongst 3MC treated mice in 12th week. Inflammation mediating leukocytes such as eosinophils, neutrophils and monocytes were also persistently apparent in the cervical infiltrations (Fig. 2Cxiii) during the entire treatment span. In the 24th and 30th weeks, the mice cervical exfoliates exhibited ‘hyperchromatic nuclei’ (Fig. 2Cxiv; indicated by arrows), assumed up a ‘bizarre-structure’ and harboured ‘perinuclear halo’ (Fig. 2Cxv, indicated by arrows) as signs of ‘vacuolation’. Cellular distortions of these types were clearly absent in PET treated groups. Acquired observations were comparable with control animals.

Noticeable cytopathological alterations in the cervix necessitated in exploration of its histology which vividly exhibited transfigurations as revealed from the presence of mild to severe cervical dysplasia (Fig. 2Diii & iv) in mice exposed to 3MC for 6 and 12 weeks. Interestingly, development of ‘invasive carcinoma’ eventuated with extension of treatment upto 16 weeks (Fig. 2Dv). Furthering 3MC treatment to 24 weeks and beyond resulted in disorientation of cervical epithelial layers (Fig. 2Dvi). As clearly comprehensible from the micrographs, no such remodelled cervical tissue features were discerned amongst the vehicle control and control mice even after 24 weeks (Fig. 2Di & ii). These aforementioned changes were in coherence with attainment of a cervical tumor. The rate of incidences of the attained dysplastic changes and tumor among Group-III mice is clearly shown in Table 2B. Moreover, chronic 3MC treatment affected the body weight of mice. The findings tabulated in Table 2A depicted a gradual loss in bodyweight of these mice in 6weeks of 3MC treatment which continued till 12 weeks (25 ± 0.1716 gms). A significant weight reduction by 24 ± 0.0100gms was observed in the 16th week which further increased by 29.1067 ± 0.1940gms in 24th week and beyond. Therefore, the decreasing trend in body weight got violated by a consistent rise in the 30th week of 3MC treatment.

Table2A: Impact of 3MC treatment upon body weight of mice.

6 weeks			12 weeks	16 weeks	24 weeks	30weeks
ANIMAL GROUPS (Weight in gms; n=10)	Group-I (Control)	25±0.2108	28±0.098	32±0.1702	33±0.16552	33.8±0.0954
	Group-II (Vehicle Control)	25.02±0.2213	27±0.1658	27.56±0.114	31.11±0.0793	32.7±0.0591
	Group-III (Treatment)	25.2±0.1079	25.8±0.171	24±0.0100	29.1067±0.194	35.4±0.09406

Table2B: Percentage (%) of 3MC treated mice displaying cervical dysplasia and tumor stages.

Normal

Mild

Dysplasia

Moderate

Dysplasia

Invasive

Carcinoma

In Situ

Anaplastic

Tumor

0^th week

(n=10)

9/10

(90%)

6^th week

(n=10)

2/10

(20%)

8/10

(80%)

12^th week

(n=10)

1/10

(10%)

2/10

(20%)

7/10

(70%)

16^th week

(n=10)

1/10

(10%)

1/10

(10%)

8/10

(80%)

24^th week

(n=10)

2/10

(20%)

8/10

(80%)

30^th week

(n=10)

1/10

(10%)

3/10

(30%)

6/10

(60%)

Chronic 3MC treatment rendered persistent cervical inflammation

3MC triggered chronic cervical inflammation in mice, as unveiled from the foregoing cytopathological excerpts demanded a thorough investigation through its nature. Enumerated DLC (%) values for Group I, II and III mice as displayed graphically in Fig. 3A-C revealed a dominance of neutrophils over other leukocytes in the mice cervix upon continual 3MC exposure. Eosinophils in the 6th week and 12th week of 3MC treatment had respective DLC values of 25.11 ± 0.58% and 35.01 ± 0.58% that underwent a little negative deviation by about 32.30 ± 0.58% in the succeeding 16th week. Conversely, neutrophils of diverse morphologies (Fig. 3E), with relatively higher DLC values (16th week: 52.13 ± 1.00%; 24th week: 66.33 ± 1.00%; 30th week: 78.01 ± 0.58%) gradually replaced the eosinophilic multitude in the cervix. Subsequently, with increasing treatment duration monocytes also joined the cervical neutrophil milieu for maintenance of the 3MC induced inflammation which could have possibly redesigned the cervical tissue architecture. Macrophages, although sparsely noted, was also obtained in the cervical infiltrates of these mice. An anecdote of cervical leukocyte infiltration trends among Group-III mice is very well-depicted in Fig. 3C. In Group-II mice, transient neutrophil infiltration was apparent only at 16th week of PET treatment (Fig. 3B). Such a short-lived inflammation was not efficacious enough to eventuate in cervical neoplasia. The observations were comparable with that of control mice from Group-I. Micrographic snippets of the varied cervical leukocytic subpopulations comprising of eosinophils, neutrophils and monocytes among 3MC treated mice are vividly portrayed in Fig. 3D.

Incessant cervical inflammation was accompanied by development of squamous cell carcinoma

Following overall analysis of cervical histology among 3MC treated groups; the cervical tumor which emerged particularly at or after 24 weeks of 3MC administration was respectively regionalized into ‘cortical’, ‘cortico-medullary’ and ‘medullary’ regions for histopathological characterization. Histological exhibits of tumor cortex (Fig. 4, indicated by arrows) hinted towards a loss of tissue organization owing to the presence of large keratin pearls. On delving into the cortico-medullary region, distortion of squamous epithelial cells (indicated by arrows) was remarkably noted. Squamous epithelial cells abnormally transformed into ‘giant tumor cells’ (indicated by arrows) harbouring aggregation of multiple nuclei in their centre (Fig. 4, lower left panel). A glimpse of the medullary tumor core, furthermore unravelled the presence of epithelial regions (indicated by arrows) which might have invaded into the stroma from the overlying tissue tier (Fig. 4, lower right panel). These histopathological findings annotated for the development of an aggressive and poorly differentiated ‘anaplastic squamous cell carcinoma’ at the mice cervix due to chronic inflammation produced by 30 weeks of 3MC treatment.

Role of inflammogenic mediator COX2 in 3MC induced carcinogenic changes

Conditions of ‘spleen’ were thoroughly studied for identifying the systemic source of leukocytes augmenting cervical inflammation. Comparative overview of the splenic anatomy and histology is well depicted in Fig. 5Ai-iv. Splenomegaly was deciphered in 3MC treated mice at and after 16th week. Corresponding splenic histopathology (Fig. 5Aiii & iv) exhibited profuse eosinophil infiltrations (indicated by arrows), along with the prevalence of an increased number of ‘germinal centres’ with intense eosin stained peripheries (indicated by arrows). These affirmed accelerating rates of leucopoiesis which primarily got incited in the mice body for combating 3MC induced cervical toxicity. However, dysregulated inflammation antagonistically paved a way for cervical carcinogenesis by further activating inflammogens. Spleen tissues isolated from vehicle control and untreated mice at 16th week were devoid of such histological remodelling.

The reproductive tissues isolated from these mice batches appeared to be strangled with blood vessels (Fig. 5B; indicated by arrows). This kind of vasculogenic changes are product of carcinogenesis-enabling vehement inflammatory reactions, which thereafter prompted for the subsequent exploration of the responsible molecular interactome involving COX2. According to western blot results (Fig. 5D), the cervical COX2 expression in mice heightened along with 3MC treatment extension. An elevated COX2 level in the tumor core was characteristically noted from the study of differential COX2 expression patterns in various tumor regions (Cortex-Tumor_COR; Cortico-medullary- Tumor_COR/MED; Medulla- Tumor_MED). Further assessment of the functional status of COX2 was performed by IHC wherein COX2 was found to be spatially distributed in the brown stained nucleus and cytoplasm (Fig. 5C) of the cervical tissue sections obtained from 3MC treated mice in the 16th and 24th weeks. A high intensity staining of about 60.44% (6/10 mice) and 72.69% (7/10 mice) were scored amongst these respective groups (Fig. 5D).

These findings endorsed an increasing trend in cervical COX2 activity (Fig. 5E). A significantly escalated COX2 activity (12th week: 3.8 ± 0.0091 U/mg; 16th week: 5.72 ± 0.016 U/mg) in mice cervix continued to rise consistently even in 24th and 30th weeks of 3MC treatment. This cumulative increase in expression and activity of COX2 protein was found to be very strongly correlated (r = 0.9750; p < 0.005) among the 3MC treated groups (Table 3A). COX2 activities among vehicle control mice were at par with the control data.

Table3(A): Pearson’s correlation coefficient among COX2 expression(COX2exp) profiles with COX2 activity, NFκB (p50), NFκB (p65), GM-CSF1, XIAP, survivin and p53 expression profiles of mice cervix subjected to chronic 3MC treatment for 0, 6, 12, 16, 24 and 30 weeks.

	COX2 exp	COX2 activity	NFκB (p50)	NFκB (p65)	GM-CSF1	XIAP	Survivin	p53
COX2 exp	1.000
COX2 activity	0.9750	1.000
NFκB (p50)	0.9550	0.9444	1.000
NFκB (p65)	0.9815	0.9280	0.9603	1.000
GM-CSF1	0.9455	0.9109	0.8742	0.9305	1.000
XIAP	0.9010	0.8570	0.9718	0.9491	0.8500	1.000
Survivin	0.9271	0.8933	0.9849	0.9642	0.8617	0.9957	1.000
p53	-0.7901	-0.7612	-0.8807	-0.912	-0.7221	-0.9200	-0.9023	1.000

Inflammation mediated COX2 activation propagated into subsequent overexpression of NFκB, GM-CSF1 and Ki67

A gain in expression of NFκB, GM-CSF1 and Ki67 (Fig. 6A) as obtained from western blot results delineated the pathway taken by activated COX2 for promoting carcinogenesis. To establish the possibility of COX2 being the culprit in upregulating NFκB (p50/p65), GM-CSF1 and Ki67, correlation coefficient were calculated and represented in a tabulated format (Table 3A). Evidentially, a strong positive correlation between each of these parameters was mathematically obtained which further strengthened the notion.

An earlier experimental observation attained by the team with the same model revealed the presence of an upregulated cervical IL6 and IL8 activities alongside high expressions [37]. These are also the upstream activators of COX2 that fuel a plethora of prosurvival signalling molecules. Consequentially, with further study elevated Granulocyte Macrophage Colony Stimulating Factor1 (GM-CSF1) expressions as evident from the incremental band intensity patterns (Fig. 6A, lower panel), corroborated with the frequent flux of neutrophils in the cervix (Fig. 3C). This justified the conviction that IL6, IL8 and GM-CSF1 cytokines along with an upregulated COX2 resulted in escalation of NFκB expressions (Fig. 6A, upper panel). In alliance with COX2 expressions, NFκB (p50/p60) subunits were observed to be very frequently localized within the nucleus at the tissue level (Fig. 6B, upper panel). In the micrographic excerpts of IHC tissue sections sampled from mice subjected to 16 and 24 weeks of 3MC treatment, positive nuclear staining for both p65 and p50 subunits of the protein was attained. High intensity positive nuclear staining for p50 and p65 enumerating to 78.77% (8/10 mice) and 82.03% (8/10 mice) were scored amongst the 16th week mice groups (Fig. 6B, lower panel). Respective protein expression scores stoking to about 80.23% (8/10 mice) and 86.23% (8/10 mice) were further quantitated amongst mice in 24 weeks of 3MC treatment (Fig. 6B, lower panel). Moreover, Ki67 expression patterns in the whole tissue proteins were fairly high (Fig. 6A, upper panel). Such a distinctively active functional status of the protein was absent amongst vehicle control and control mice during this interval.

Chronic 3MC treatment induced COX2 enabled IAP activations

In order to assess the strength of this prosurvival signalling in overriding apoptotic cues the disposition of XIAP and survivin was analysed in the cervical tissues of 3MC treated mice. Western blot results as showcased in Fig. 7A clearly indicated the rising patterns in protein expressions of XIAP and survivin in the mice cervical tissue lysates at the treatment interims. Particularly, for XIAP, a hike in expressions was attained at 16th week of 3MC treatment. Survivin, on the contrary was not as high as XIAP while in relation to control and vehicle control mice the expression profiles was considerably high.

IHC results as portrayed in Fig. 7B exposed the presence of functional XIAP and survivin proteins in the cellular cytoplasm of cervical tissue sections specifically obtained from 3MC treated mice in 16th and 24th weeks. Respective positive staining intensities for XIAP scored to about 75.25 %(~ 7/10mice) and 78.625 %(~ 8/10mice) were documented amongst these respective batches. Similarly, the cytoplasm was comparatively less stained (16weeks:55%; 5/10mice, 24weeks:59.98%; ~6/10mice) for survivin. Again, XIAP (r = 0.8570 & r = 0.9010; p < 0.005) and survivin (r = 0.8933& r = 0.9271; p < 0.005) expressions were in strong positive correlation with the activity and expressions of COX2 in the 3MC treated mice (Table 3A).Absence of these findings in the vehicle control and control animals were imperative of the involvement of NFκB /COX2/IAP axis in development of cervical neoplasia in 3MC treated mice.

COX2 activation immortalized cervical cells by facilitating Ki67 and PCNA expressions

The spatial distribution of proliferative antigens Ki67 and PCNA was studied by IHC in order to assess the proliferative potentials of the cervical epithelium as a culmination of NFκB /COX2/IAP functions. Positive staining of cellular nucleus in all the differential cervical epithelial layers was noted amongst the tissue sections of mice subjected to 3MC treatment for 16 and 24 weeks or more (Fig. 8A). These patterns were delimited only to the undifferentiated proliferative basal epithelial cell layers of the control and vehicle control groups. Clear manifestations of uniform staining for Ki67 and PCNA of all cells in the cervical epithelium were scored and illustrated comparatively in Fig. 8B.

Inflammation induced systemic stress generates free radicals by disrupting antioxidant defence mechanism

To further find out the systemic impact of chronic cervical 3MC treatment in mice, ROS and RNS levels were quantified in the blood leukocytes and peritoneal macrophages. Relative interpretations of these findings were undertaken with respect to iNOS and the total antioxidant enzyme activities. A significant appraisal in ROS levels were noted in 3MC treated mice from 16th week onwards (Fig. 9A). These observations were coherent with that of RNS levels among these same mice groups (Fig. 9B). Nitrite generation was documented to rise from 16th week (3.813 ± 0.00686 µM/10⁶cells/min) which was found to attain peak by 30 weeks of 3MC treatment. Both of these findings coincided with the activity trends of iNOS as shown in Fig. 9C where the enzyme activity was found to produce citrulline of about 11.89 ± 0.15311% in the 16th week specifically. These parameters were found to be subletting each other for promoting systemic stress as revealed from their significantly strong positive correlations (Table 3B).

Table3(B): Pearson’s correlation coefficient among cervical COX2 activity and collateral systemic parametres ROS levels, RNS levels, iNOS activity and Comet Tail Moment(CTM) of mice subjected to chronic 3MC treatment for 0, 6, 12, 16, 24, and 30 weeks.

	COX2 activity	ROS	RNS	iNOS	CTM
COX2 activity	1.000
ROS	0.9872	1.000
RNS	0.9523	0.9537	1.000
iNOS	0.9648	0.9446	0.9708	1.000
CTM	0.9606	0.9456	0.9652	0.9974	1.000

All these findings necessitated to look into the activities of the total antioxidant enzyme capacities. Graphical overview clearly depicted gradual rise in free radical scavenging activity up to 12th and 16th week of treatment (Fig. 9D), which subsequently declined by 24 and 30 weeks. Relatively, in the vehicle control groups the free radical scavengers were found to function properly with exposure to PET like that of control one. The subtly increasing trend of anti-oxidant enzyme activities holistically helped to quench the PET generated free radicals which went unquenched in the carcinogen treated groups.

Cervical inflammation imposed systemic genotoxic stress eventually deregulating key tumor suppressor proteins

To ascertain the genotoxic profile upon 3MC exposure mediated inflammatory stress, comet assay was performed with the leukocytes isolated from mice blood of all the three groups. Fluorescent microscopic images exhibited the distribution patterns of damaged DNA in terms of DNA migration (comet formation) among different groups (Fig. 10Ai-xv). The results upheld the appearance of distinct DNA-tails among the 3MC treated groups with incremental time period. Among the PET treated mice, presence of such damage was indistinct just like that of the untreated animals. Concomitant rise in the comet tail moment as calculated by KOMET software is represented graphically in Fig. 10B. Results were indicative of the impact of free radical mediated stress upon the DNA (Table 3B).

Blood bearing leukocytes harbouring damaged DNA also drain into cervical region which was under eventual transformation by the hiked COX2 activities. Continual inflammation driving changes mediated by COX2 accompanied subsequent reduction in expression profiles of tumor supressors at the protein levels in the mice cervical tissue extracts. Western blot data as depicted in Fig. 10C are imperative of the gradual fall in protein expressions of p53, p21 and Rb, specifically at and after 16 weeks of 3MC treatment. Unstable expression profiles of acetylated-p53 (Lys373), the active form of the protein, further supported the observation. These inferences bridge the impact of inflamogenic stress induced in the cervix with the role of the simultaneously induced systemic stress in favouring cervical carcinogenesis in this stud

In spite of therapeutic advancements, higher expression status of COX2 in cervical cancer surmounts in therapy resistance followed by disease relapse. Traditionally being a HPV mediated cancer the viral oncoproteins E5, E6 and E7 were found to turn on ‘amphiregulin’, the COX2 activator either in a ligand dependent or independent manner via MAPK or PI3K/Akt pathways [42, 43]. In coherence with HPV mediated cervical cancers, the present model also exhibited an escalated COX2 kinetics. Hence, the role of COX2 as an orchestrator of dysregulated inflammation in inducing cervical neoplastic changes upon chronic treatment with 3MC is well highlighted in this study. Concomitant rise in expression and activity of COX2 accelerated expressions of prosurvival molecules like NFκB (p50/p65), GM-CSF1, XIAP and survivin within the mice cervix tissue with prolongation of treatment duration. This clearly annotated for the presence of a ‘positive feedback loop’ of prosurvival signalling molecules which paved a way for cervical neoplasia (Table 3A).

The current results along with previous laboratory findings of elevated expressions and activities of inflammatory cytokines IL6 and IL8 in the cervical tumor microenvironment of 3MC treated mice favouring carcinogenic progression, were in accordance with the study of Li et al (2018) wherein IL6, IL8 and COX2 triggered each other to bring about carcinogenesis [6]. The present study confirmed localization of COX2 protein in the nucleus as well as cytoplasm of the cervical tissues obtained from mice undergoing 3MC treatment, predominantly for 16 weeks, 24 weeks and onwards. This hinted towards the prevalence of an upregulated COX2 dynamics in the cervix. Quantitative estimation of COX2 activity also reflected a stimulated trend in enzyme kinetics. Such an inference enabled identification of COX2 as the protagonist molecule directing this episode of ‘inflammogenesis of cervical cancer’.

Further experimentation revealed convenient regulation by COX2 in generation of a molecular interactome involving erratically expressed tumor suppressor proteins (p53, p21 and Rb) and upregulated prosurvival proteins which majorly included NFκB, XIAP and survivin in 3MC driven cervical carcinogenesis. This eventually rendered an impact in transformation of the reproductive organ anatomy, cytopathology, histopathology and biochemistry of the carcinogen treated mice. COX2 and p53 are antagonistic in function because the latter is known to positively regulate ‘thrombospondin-1’ which directly inhibits angiogenesis by shutting down the angiogenic effector molecule, VEGF [28, 29]. Hypoxia induced functional facilitation of p53 is a common occurrence within a tumor lump, alongside activation of NFκB and COX2. However, during carcinogenic discourse, growth aiding cues received in terms of hiked iNOS activity was reported to encourage NFκB and COX2 for overriding the p53 action so as to progress with neoplasia [44, 45]. In agreement with these previously published reports, the tumor obtained after 24 weeks of carcinogen treatment in this present study also exhibited a high COX2 and NFκB expressions in the tumor core with concomitantly high COX2 activity. IHC results fostering nuclear accumulation of p65 and p50 subunits of NFκB was indicative of its active functional stature. Nonetheless, an eventual decline in p53 correspondingly accompanied by rise in IAPs with time was an alibi to COX2 being the silent umpire of the interplay between these promoters of carcinogenesis.

Another interesting observation of the present study was formation of enlarged spleen (splenomegaly) which was specifically observed in animals at 16th week of 3MC treatment and onwards. Documentation of the cumulative experimental findings had unveiled the treatment period of 16th week as the ‘crucial-interim’ for the development of invasive cancer among these mice. Enlarged spleen showcased an altered histopathology which was characterized by the presence of eosinophil inflitrations and enhanced number of ‘germinal centres’; the hubs which nourish and train the naïve blood leukocytes to become immunologically competent [46]. Continual carcinogenic treatment in mice for 30weeks caused outbursts of free radicals to mediate systemic stress as evident from escalated iNOS activity, indiscriminately increased ROS and RNS levels which conjugatively compromised scavenging capacity of the antioxidant enzymes. Impact of these multiple oxidative hits was apparent in the form of concurrent damage incurred upon the leukocyte DNA as observed from Comet Assay (Table 3B). Being a PAH, 3MC gets potentiated as a carcinogen only after biotransformation. Thus, its effect upon the systemic parameters in the long run was inevitable. Moreover, COX2 was also reported to promote invasion of cervical cancer cells into lymphoid organs and blood vasculature [47]. In all probability, causation of splenomegaly was also one of the attributions of COX2 in this experimental model

Cervix is under constant immune surveillance since it is always patrolled by leukocytes, specifically eosinophils besides having an acidic niche. Any shift or drift in the cervical leucocyte subpopulation is therefore alarming. In this study, a shoot up of neutrophil with altered morphologies accompanied by monocytes gradually replaced the eosinophilic multitude of the mice cervix at 16 weeks of 3MC treatment which persisted with time to quench the carcinogenic assault. These inflamogenic mediators seem to have drained into the cervix following seepage from an enlarged spleen as relentless leucopoiesis was reported to render splenomegaly [48]. Molecular insights further evinced a parallel rise in GM-CSF1, the key functional neutrophil differentiator. Evidentially, COX2 in this study acted as a driver of the cervical inflammatory events which eventually amplified into the entire system of the animals.

XIAP and survivin are vital prosurvival molecules which migrate from the nucleus to the cytoplasm for becoming functionally active. Cytosolic IAPs bind to the caspase enzymes, the cellular executioners, thereby rendering them inactive [49]. In the present study, appreciable cytosolic dislocation of the respective IAPs on and after 16th week of treatment span was obvious. This was convoyed by collateral nuclear accumulation of the proliferative antigens, Ki67 and PCNA. As comprehendible from immunostaining of cervix tissues for Ki67 and PCNA (IHC results), the immortalized cells of cervix were found to be expressing these proliferative antigens which subsequently disbursed from the proliferative basal layer to rest of the differential cervical epithelial layers. COX2 mediated activation of prosurvival effectors for boosting malignant cervical transformations could be vividly envisioned from this present finding. Upregulated COX2 with all its allies was tailoring the never ending saga of 3MC induced carcinogenic inflammation to enable the development of cervical carcinoma. Overall findings have been schematically represented in Fig. 11.

The in vivo model established here flaunts a microenvironment which has upregulated prosurvival molecules under the command of COX2. Thus, it can be treated as a model to address issues of therapy resistance that stymies cervical cancer treatment. This referral model throws paramount light upon ways to target COX2 and its several unknown effectors as prognostic and diagnostic biomarkers for planning a better treatment rationale.

AUTHOR CONTRIBUTION

Elizabeth Mahapatra has predominantly acquired all the data, analysed them to design the concept for this paper and has accordingly composed the manuscript with assistance from Souvick Biswas and Salini Das.

Sutapa Mukherjee has designed the project work and has critically checked through the manuscript.

Madhumita Roy has checked through the manuscript.

FUNDING

The authors thankfully acknowledge Ministry of Health and Family Welfare, Govt. of India for providing financial aid through Intramural Project Funding being earned by Dr. Sutapa Mukherjee (Senior Scientific Officer-I, Dept. of Environmental Carcinogenesis & Toxicology, CNCI, Kolkata, India).

ACKNOWLEDGEMENT

The authors are indebted to Director, CNCI for providing infrastructural facilities. The authors heartily thank Dr. Debasish Chakraborty (Assistant Professor, Dept. of Pathology, IPGMER, Kolkata, India) for helping them in analysis of the histology results and Dr. Abhijit Rakshit (Head of Department, Dept. of Animal Care and Maintenance, CNCI, Kolkata, India) for his invaluable inputs in animal segregation and batching. With special mention, the authors humbly acknowledge the contribution of Dr. Chinmay Kumar Panda (Ex-Head of Department, Dept. of Oncogene Regulation Unit, CNCI, Kolkata, India), for guidance in identification of the correct time-period of this experimental study.

DATA AVAILABILITY

All the datasets displayed in the current study are available from the corresponding author on reasonable request.

CODE AVAILABILITY

There is no code available.

Conflict of Interest. The authors declare that they have no known conflicts of interest.

Ethics Approval. All animal experimentations were carried out in accordance with IAEC ethical norms. Mice were procured only after ethical clearance (IAEC Proposal No# IAEC-1774/SM-1/2018/7) for the concerned project.

Consent to participate. All authors have wilfully participated in the concerned work following which they have given complete consent for publication.

Consent for Publications. All authors have given their consent for this publication.

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COX2: A Prognostic Inflammogenic Marker Drives Cervical Carcinogenesis In Vivo Through NFκB/IAP/p53Axis

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Abstract

Figures

Introduction

Material And Methods

Animal Maintenance

Experimental design

Record of body weight and carcinogenic incidences

Cytopathological study

Enumeration of differential count of cervical leukocytes

Histology

Immunohistochemistry

Preparation of tissue lysates

Estimation of total protein

Western Blot Analysis

Quantitative estimation of COX2 activity

Isolation of blood leukocytes

ROS generation

RNS generation

Quantitative estimation of nitric oxide synthetase activity

Antioxidant Enzyme Activity

Single cell gel electrophoresis (SCGE or Comet assay)

Statistical Analysis

Results

Emergence of tumor at the cervical region upon chronic 3MC treatment

Chronic 3MC treatment rendered persistent cervical inflammation

Incessant cervical inflammation was accompanied by development of squamous cell carcinoma

Role of inflammogenic mediator COX2 in 3MC induced carcinogenic changes

Inflammation mediated COX2 activation propagated into subsequent overexpression of NFκB, GM-CSF1 and Ki67

Chronic 3MC treatment induced COX2 enabled IAP activations

COX2 activation immortalized cervical cells by facilitating Ki67 and PCNA expressions

Inflammation induced systemic stress generates free radicals by disrupting antioxidant defence mechanism

Cervical inflammation imposed systemic genotoxic stress eventually deregulating key tumor suppressor proteins

Discussion

Conclusion

Declarations

References

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Version 1